m5 cytokines cocktail  (R&D Systems)

Journal: bioRxiv

Article Title: PEPITEM Tripeptides and Peptidomimetics: Next-Generation Modulators of Inflammation in Immune-Mediated Conditions

doi: 10.1101/2024.10.14.618151

Figure Lengend Snippet: The effect of PEPITEM and SVT[NH-Ethyl] is mirrored in the lymphoid organ repertoire. A) Spleen weight/mouse body weight ratio (expressed as g) evaluated at the experimental endpoint (day 7). Flow cytometry analysis of splenic B) CD4 + , C) CD8 + , D) Th1 (CD4 + IFN-γ + ) and E) Th17 (CD4 + IL-17 + ) subsets, pre-gated on living cells (see flow cytometry strategy reported in Supplementary Fig. 2B and C). Histograms indicate the total positive populations (expressed as x10 6 ) in the different experimental conditions. F) Quantification of T-related cytokines performed by a multi-LEGENDplex ™ analyte flow array (gating strategy is reported in Supplementary Fig. 3) on lymphocytes isolated from skin-draining lymph nodes, following ex vivo stimulation with PMA and ionomycin, presented as a heatmap (colormap:double gradient) and expressed as pg mL −1 . G), H) and I) TNF-α, IL-6 and IL-17A cytokines extrapolated from the heatmap and represented graphically as histograms. Data are presented as mean ± S.D. of n=6 animals in each group. Statistical analysis was conducted by one- or two-way ANOVA followed by Bonferroni’s or Dunnett’s posthoc-test. ## P ≤ 0.01, ### P ≤ 0.001, #### P ≤ 0.0001 vs CTRL group; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 vs Imiquimod group.

Article Snippet: Psoriasis-like keratinocytes model was established by stimulating HaCaT cells with M5 cytokines cocktail consisting of 2.5 ng mL −1 of each TNF-α (210-TA/CF), Oncostatin M (295-OM/CF), IL-1α (200_LA/CF), IL-17 (317-ILB) and IL-22 (782-IL/CF, all from R&D System, Italy) as previously described , .

Techniques: Flow Cytometry, Isolation, Ex Vivo

Journal: bioRxiv

Article Title: PEPITEM Tripeptides and Peptidomimetics: Next-Generation Modulators of Inflammation in Immune-Mediated Conditions

doi: 10.1101/2024.10.14.618151

Figure Lengend Snippet: A) and B) In vitro mechanistic studies on PEPITEM, tripeptides and peptidomimetics. In vitro cytotoxic examination, evaluated by MTT assay, for PEPITEM, tripeptides and peptidomimetics performed on J774A.1 murine macrophage cell lines, following 4 and 24 h of treatment with the selected concentrations (1-30 ng mL −1 ). Dotted lines indicate 75% of cell viability. Data are expressed as cell viability (% of control) and presented as mean ± S.D. of 3 independent experiments. C) and D) IL-6 and TNF-α ELISA assays performed on supernatant of J774A.1 pre-treated with indicated compounds at the concentration of 10 ng mL −1 (highest non-cytotoxic concentration) and then stimulated with LPS (10 µg mL −1 ) for 24 h. E) and F) IL-6 and TNF-α ELISA assays performed on supernatants of NIH-3T3 mouse fibroblasts pre-treated with indicated compounds at the concentration of 10 ng mL −1 and then stimulated with LPS (10 µg mL −1 ) for 24 h. ( C-F ) Data are expressed as pg mL −1 and presented as means ± S.D. of 3 independent experiments. Statistical analysis was conducted by one-way ANOVA followed by Bonferroni’s posthoc-test. # P ≤ 0.05, ### P ≤ 0.001, #### P ≤ 0.0001 vs CTRL group; *P ≤ 0.05, **P ≤ 0.01 vs LPS group. G) Effect of tested compounds on hyperproliferation of M5 cytokines (TNF-α, Oncostatin M, IL-1α, IL-17 and IL-22)-stimulated HaCaT cells, as in vitro psoriasis-like model. HaCaT hyperproliferation (expressed as cells proliferation, % of control) was measured using MTT assay and presented as mean ± S.D. of 3 independent experiments. Statistical analysis was conducted by one-way ANOVA followed by Bonferroni’s posthoc-test. *P ≤ 0.05 vs M5 group.

Article Snippet: Psoriasis-like keratinocytes model was established by stimulating HaCaT cells with M5 cytokines cocktail consisting of 2.5 ng mL −1 of each TNF-α (210-TA/CF), Oncostatin M (295-OM/CF), IL-1α (200_LA/CF), IL-17 (317-ILB) and IL-22 (782-IL/CF, all from R&D System, Italy) as previously described , .

Techniques: In Vitro, MTT Assay, Control, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Clinical, Cosmetic and Investigational Dermatology

Article Title: Inhibition of Key Glycolytic Enzyme Hexokinase 2 Ameliorates Psoriasiform Inflammation in vitro and in vivo

doi: 10.2147/CCID.S435624

Figure Lengend Snippet: 3-BrPA suppresses M5-stimulated HaCaT cell proliferation and glycolysis. ( A ) The relative expression levels of HK2 RNA and protein in M5 stimulated HaCaT cells and control cells. ( B ) The proliferation and glycolysis in M5 stimulated HaCaT cells and control cells. ( C ) The cell proliferation, glucose consumption and lactate production in 3-BrPA treated cells and control cells. ( D ) Regulation of the G1/S transition in 3-BrPA treated cells and control cells. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: HaCaT cells were treated with a cocktail of cytokines, referred to as M5, consisting of interleukin (IL)-17A, tumor necrosis factor-alpha (TNF-α), IL-1α, IL-22, and oncostatin-M at a final concentration of 10 ng/mL (Peprotech), to induce psoriatic inflammation.

Techniques: Expressing, Control